RESUMO
BACKGROUND: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity. METHODS: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks. RESULTS: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1ß expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes. CONCLUSION: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Hipertensão , Fator 2 Relacionado a NF-E2 , Núcleo Hipotalâmico Paraventricular , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Ribonucleotídeos , Transdução de Sinais , Animais , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Masculino , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Aminoimidazol Carboxamida/administração & dosagem , Ribonucleotídeos/farmacologia , Ribonucleotídeos/administração & dosagem , Proteínas Quinases Ativadas por AMP/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Citocinas/metabolismoRESUMO
OBJECTIVE: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. METHODS: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and ß subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a ß3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. RESULTS: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and ß2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and ß2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. CONCLUSIONS: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for ß3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glicemia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Tecido Adiposo Marrom/metabolismo , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Animais , Benzimidazóis/administração & dosagem , Dieta Hiperlipídica , Feminino , Teste de Tolerância a Glucose , Insulina/metabolismo , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Animais , Piridinas/administração & dosagem , Ribonucleotídeos/administração & dosagem , Termogênese/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the efficacy and safety of the AMPK activator AICAR alone or in combination with decitabine on myelodysplastic syndromes (MDS). RESULTS: p-AMPK (Thr172) expression was lower in MDS samples than in healthy donors. AMPK agonist AICAR inhibited the proliferation of MDS cell lines (SKM1 and MDS-L) (P < 0.05). The results from flow cytometry suggested that AICAR induced G0/G1 phase arrest and apoptosis through inducing DNA damage, as confirmed by immunofluorescence analysis in MDS cell lines. AICAR alone or in combination with decitabine was applied to the two MDS cell lines, and the combination index values at all concentrations were significantly < 1. This strong synergistic effect was also corroborated in the primary MDS patient samples and in an MDS cell line xenograft mouse model. Furthermore, immunohistochemical staining showed that there was more DNA damage accumulation in the combination group than that in any other groups. CONCLUSION: This is the first report on how the AICAR suppresses MDS cell proliferation and synergizes with decitabine via DNA damage induction. AICAR in combination with decitabine may be a promising therapeutic strategy in MDS.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Dano ao DNA , Decitabina/administração & dosagem , Síndromes Mielodisplásicas/tratamento farmacológico , Ribonucleotídeos/administração & dosagem , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Decitabina/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Ribonucleotídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The fungicide Iprodione is widely applied in vegetables and raises concern for human health. The A549 human lung carcinoma cell line is a suitable model for assessing the toxicological effects of drugs. The goal of this work was to evaluate the genotoxicity and oxidative stress in the A549 cell line exposed to sublethal concentrations from 3 to 100 µg/mL Iprodione considering LC50 = 243.4 µg/mL Iprodione, as determined by the MTT assay. Generalized Linear Mixed Models (GLMM) were performed to determine the association between the responses NDI, MNim and MNib and the explanatory variables. Iprodione and solvent were relativized to the control whereas the concentration was included as numeric variable. ANOVA was used for the comparison of treatments. The coefficients of linear association between the explanatory variables and NDI, and the coefficients of logistic association between explanatory variables and MNim were not significant. However, these coefficients showed significant association with MNib only for Iprodione treatment but not for Iprodione concentration, indicating lack of dose-response relationship. Genotoxicity risk assessment indicated that the increase in Iprodione concentrations increased slightly the probability of belonging to the genotoxic category. ANOVA showed significant differences in MNib, and non-significant differences in NDI and MNim among treatments. The oxidative stress analysis performed at 3, 12, and 25 µg/mL Iprodione showed a significant and linear increase in SOD, and a significant and linear decrease in GSH and GST. The Dunnett test was significant for GSH at 12 and SOD at 25 µg/mL.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Fungicidas Industriais/toxicidade , Hidantoínas/toxicidade , Mutagênicos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Células A549 , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/toxicidade , Relação Dose-Resposta a Droga , Fungicidas Industriais/administração & dosagem , Humanos , Hidantoínas/administração & dosagem , Dose Letal Mediana , Neoplasias Pulmonares/metabolismo , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Medição de Risco , Superóxido Dismutase/metabolismoRESUMO
A novel nanocomposite poly(ethylene-co-vinyl acetate) (EVA) film with controlled in vitro release of iprodione (ID) was prepared. Chitosan (CS) was used as the reinforcement which enhances the water and oxygen permeability of films. ID loaded poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) (IPP) micelles were used as the drug carrier which endows the films with antifungal and controlled release ability. IPP micelles with spherical shape and uniform size were obtained, and the maximum encapsulation efficacy (EE) was 91.17 ± 5.03% by well controlling the feeding amount of ID. Incorporation CS could improve the oxygen and moisture permeability of films, and the maximum oxygen permeability (OP) and water vapor transmission rate (WVTR) were 477.84 ± 13.03 cc/(m2·d·0.1 MPa) and 8.60 ± 0.25 g m-2 d-1, respectively. After loading IPP micelles, the films showed an improved antifungal ability and temperature-sensitive drug release behavior, and were found to enhance the quality of grapes by pre-harvest spraying.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hidantoínas/farmacocinética , Nanocompostos/química , Vitis/efeitos dos fármacos , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/farmacocinética , Quitosana/química , Preparações de Ação Retardada , Portadores de Fármacos , Microbiologia de Alimentos , Fungicidas Industriais/administração & dosagem , Fungicidas Industriais/farmacocinética , Hidantoínas/administração & dosagem , Lactonas/química , Micelas , Oxigênio , Permeabilidade , Polietilenoglicóis/química , Polivinil/química , VaporRESUMO
High consumption of saturated fats links to the development of hypertension. AMP-activated protein kinase (AMPK), a nutrient-sensing signal, is involved in the pathogenesis of hypertension. We examined whether early intervention with a direct AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) during pregnancy or lactation can protect adult male offspring against hypertension programmed by high saturated fat consumption via regulation of nutrient sensing signals, nitric oxide (NO) pathway, and oxidative stress. Pregnant Sprague-Dawley rats received regular chow or high saturated fat diet (HFD) throughout pregnancy and lactation. AICAR treatment was introduced by intraperitoneal injection at 50 mg/kg twice a day for 3 weeks throughout the pregnancy period (AICAR/P) or lactation period (AICAR/L). Male offspring (n = 7-8/group) were assigned to five groups: control, HFD, AICAR/P, HFD + AICAR/L, and HFD + AICAR/P. Male offspring were killed at 16 weeks of age. HFD caused hypertension and obesity in male adult offspring, which could be prevented by AICAR therapy used either during pregnancy or lactation. As a result, we demonstrated that HFD downregulated AMPK/SIRT1/PGC-1α pathway in offspring kidneys. In contrast, AICAR therapy in pregnancy and, to a greater extent, in lactation activated AMPK signaling pathway. The beneficial effects of AICAR therapy in pregnancy is related to restoration of NO pathway. While AICAR uses in pregnancy and lactation both diminished oxidative stress induced by HFD. Our results highlighted that pharmacological AMPK activation might be a promising strategy to prevent hypertension programmed by excessive consumption of high-fat food.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Fenômenos Fisiológicos da Nutrição Animal , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/efeitos adversos , Hipertensão/etiologia , Hipertensão/prevenção & controle , Ribonucleotídeos/administração & dosagem , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/fisiologia , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/farmacologia , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Animais , Feminino , Rim/metabolismo , Lactação/metabolismo , Masculino , Óxido Nítrico/metabolismo , Obesidade/etiologia , Obesidade/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Projetos Piloto , Gravidez , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Potenciais de Ação/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Ouabaína/administração & dosagem , Ouabaína/farmacologia , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Masculino , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Ratos , Ratos Wistar , Ribonucleotídeos/administração & dosagem , Ribonucleotídeos/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Relação Estrutura-AtividadeRESUMO
AMP-activated protein kinase (AMPK) is a member of Ser/Thr kinases that has been shown to regulate energy balance. Although recent studies have demonstrated the function of AMPK in adipose tissue using different fat-specific AMPK knockout mouse models, the results were somewhat inconsistent. For this study, we tested the hypothesis that AMPK in adipose tissue regulates whole body glucose metabolism. To determine the role of adipose tissue AMPK in vivo, we generated fat-specific AMPKα1/α2 knockout mice (AMPKFKO) using the Cre-loxP system. Body weights of AMPKFKO mice were not different between 8 and 27 weeks of age. Furthermore, tissue weights (liver, kidney, muscle, heart and white and brown adipose tissue) were similar to wild type littermates and DEXA scan analysis revealed no differences in percentages of body fat and lean mass. Knockout of AMPKα1/α2 in adipose tissue abolished basal and AICAR-stimulated phosphorylation of AMPK and Acetyl-CoA Carboxylase, a downstream of AMPK. Despite of the ablation of AICAR-stimulated AMPK phosphorylation, the blood glucose-lowering effect of AICAR injection (i.p.) was normal in AMPKFKO mice. In addition, AMPKFKO displayed normal fasting blood glucose concentration, glucose tolerance, insulin tolerance, and insulin signaling, indicating normal whole body glucose metabolism. These data demonstrate that adipose tissue AMPK plays a minimum role in whole body glucose metabolism on a chow diet.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Glucose/metabolismo , Ribonucleotídeos/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/metabolismo , Animais , Dieta , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ribonucleotídeos/administração & dosagemRESUMO
OBJECTIVE: Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor whose phosphorylation increases energy production. We sought to evaluate the placenta-specific effect of AMPK activation on the handling of nutrients required for fetal development. METHODS: Explants were isolated from term placenta of 29 women (pregravid body mass index: 29.1 ± 9.9 kg/m2) and incubated for 24 hours with 0 to 100 µmol/L resveratrol or 0 to 1 mmol/L of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). Following treatment, uptake and metabolism of radiolabeled fatty acids and glucose were measured. Phosphorylation of AMPK was measured by Western blotting. Adenosine diphosphate (ATP) production was assessed using the mitochondrial ToxGlo assay kit. P < .05 was considered statistically significant. RESULTS: Resveratrol and AICAR increased AMPK phosphorylation in human placental explants. Exposure to resveratrol decreased the uptake of polyunsaturated fatty acids, arachidonic acid, and docosahexaenoic acid at 100 µmol/L ( P < .0001). Fatty acid oxidation was decreased by 100 µmol/L ( P < .05) resveratrol, while esterification was unchanged. Resveratrol decreased glucose uptake at the 50 and 100 µmol/L doses ( P < .05). Glycolysis was not significantly affected. AICAR had similar effects, decreasing fatty acid uptake and glycolysis ( P < .05). Production of ATP declined at doses found to decrease nutrient metabolism ( P < .05). CONCLUSIONS: Activation of AMPK in the human placenta leads to global downregulation of metabolism, with mitotoxicity induced at the doses of resveratrol and AICAR used to activate AMPK. Although activation of this pathway has positive metabolic effects on other tissues, in the placenta there is potential for harm, as inadequate placental delivery of critical nutrients may compromise fetal development.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Mitocôndrias/metabolismo , Placenta/metabolismo , Adulto , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Inibidores Enzimáticos/administração & dosagem , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Humanos , Fosforilação , Gravidez , Resveratrol/administração & dosagem , Ribonucleotídeos/administração & dosagem , Trofoblastos/metabolismoRESUMO
Premature baboons exhibit peripheral insulin resistance and impaired insulin signaling. 5' AMP-activated protein kinase (AMPK) activation improves insulin sensitivity by enhancing glucose uptake (via increased glucose transporter type 4 [GLUT4] translocation and activation of the extracellular signal-regulated kinase [ERK]/ atypical protein kinase C [aPKC] pathway), and increasing fatty acid oxidation (via inhibition of acetyl-CoA carboxylase 1 [ACC]), while downregulating gluconeogenesis (via induction of small heterodimer partner [SHP] and subsequent downregulation of the gluconeogenic enzymes: phosphoenolpyruvate carboxykinase [PEPCK], glucose 6-phosphatase [G6PASE], fructose- 1,6-bisphosphatase 1 [FBP1], and forkhead box protein 1 [FOXO1]). The purpose of this study was to investigate whether pharmacologic activation of AMPK with AICAR (5-aminoimidazole-4-carboximide riboside) administration improves peripheral insulin sensitivity in preterm baboons. 11 baboons were delivered prematurely at 125±2 days (67%) gestation. 5 animals were randomized to receive 5 days of continuous AICAR infusion at a dose of 0.5 mg·g-1·day-1. 6 animals were in the placebo group. Euglycemic hyperinsulinemic clamps were performed at 5±2 and 14±2 days of life. Key molecules potentially altered by AICAR (AMPK, GLUT4, ACC, PEPCK, G6PASE, FBP1, and FOXO1), and the insulin signaling molecules: insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were measured using RT-PCR and western blotting. AICAR infusion did not improve whole body insulin-stimulated glucose disposal in preterm baboons (12.8±2.4 vs 12.4±2.0 mg/(kg·min), p = 0.8, placebo vs AICAR). One animal developed complications during treatment. In skeletal muscle, AICAR infusion did not increase phosphorylation of ACC, AKT, or AMPK whereas it increased mRNA expression of ACACA (ACC), AKT, and PPARGC1A (PGC1α). In the liver, INSR, IRS1, G6PC3, AKT, PCK1, FOXO1, and FBP1 were unchanged, whereas PPARGC1A mRNA expression increased after AICAR infusion. This study provides evidence that AICAR does not improve insulin sensitivity in premature euglycemic baboons, and may have adverse effects.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/administração & dosagem , Resistência à Insulina , Insulina/metabolismo , Ribonucleotídeos/administração & dosagem , Administração Intravenosa , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/sangue , Animais , Animais Recém-Nascidos , Ácidos Graxos não Esterificados/sangue , Feminino , Glicogênio/sangue , Hipoglicemiantes/sangue , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Papio , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ribonucleotídeos/sangueRESUMO
Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Distrofia Miotônica/tratamento farmacológico , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Ribonucleotídeos/administração & dosagem , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Mioblastos/efeitos dos fármacos , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Resveratrol/administração & dosagem , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Insulin degrading enzyme (IDE) is believed to act as a junction point of Type 2 diabetes (T2D) and Alzheimer's disease (AD); however, the underlying mechanism was not completely clear yet. Transgenic APPSwe/PS1 mice were used as the AD model and were treated with streptozocin/streptozotocin (STZ) to develop a mixed mice model presenting both AD and T2D. Morris Water Maze (MWM) and recognition task were performed to trace the cognitive function. The detection of fasting plasma glucose (FPG) and plasma insulin concentration, and oral glucose tolerance test (OGTT) were used to trace the metabolism evolution. Aß40 and Aß42 were quantified by colorimetric ELISA kits. The mRNA or protein expression levels were determined by quantitative real-time RT-PCR and Western blotting analysis respectively. T2D contributes to the AD progress by accelerating and worsening spatial learning and recognition impairments. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The expression levels of IDE, PPARγ, and AMPK were down-regulated in mice with AD and T2D. PPARγ activator rosiglitazone (RSZ) or AMPK activator AICAR increased the expression level of IDE and decreased Aß levels in mice with AD and T2D. RSZ or AICAR treatment also alleviated the spatial learning and recognition impairments in AD and T2D mice. Our results found that, in the mice with T2D and AD, the activators of PPARγ/AMPK signaling pathway significantly increased the expression level of IDE, and decreased the accumulation of Aß40 and Aß42, as well as alleviated the spatial learning and recognition impairments.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Doença de Alzheimer/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Insulina/metabolismo , Insulisina/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Peptídeos beta-Amiloides/metabolismo , Animais , Glicemia , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Jejum , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Humanos , Insulina/genética , Aprendizagem/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , PPAR gama/genética , Ribonucleotídeos/administração & dosagem , Rosiglitazona , Estreptozocina/toxicidade , Tiazolidinedionas/administração & dosagemRESUMO
Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637 cells in a time- and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzyme A carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compound C partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compound C had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.
Assuntos
Autofagia/efeitos dos fármacos , Benzilisoquinolinas/administração & dosagem , Proteínas Quinases/genética , Serina-Treonina Quinases TOR/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Quinases/efeitos dos fármacos , Ribonucleotídeos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
SUR2A is an 'atypical' ABC protein that forms sarcolemmal ATP-sensitive K+ (KATP ) channels by binding to inward rectifier Kir6.2. Manipulation with SUR2A levels has been suggested to be a promising therapeutic strategy against ischaemic heart diseases and other diseases where increased heart resistance to stress is beneficial. Some years ago, it has been reported that high-altitude residents have lower mortality rates for ischaemic heart disease. The purpose of this study was to determine whether SUR2A is regulated by mild-to-severe hypoxic conditions (15% oxygen; oxygen tension equivalent to 3000 m above sea level) and elucidate the underlying mechanism. Mice were exposed to either to 21% (control) or 15% concentration of oxygen for 24 hrs. Twenty-four hours long exposure to 15% oxygen decreased partial pressure of O2 (PO2 ), but did not affect blood CO2 (PCO2 ), haematocrit nor levels of ATP, lactate and NAD+/NADH in the heart. Cardiac SUR2A levels were significantly increased while Kir6.2 levels were not affected. Hypoxia did not induce phosphorylation of extracellular signal-regulated kinases (ERK1/2) or protein kinase B (Akt), but triggered phosphorylation of AMP activated protein kinase (AMPK). AICAR, an activator of AMPK, increased the level of SUR2A in H9c2 cells. We conclude that oxygen increases SUR2A level by activating AMPK. This is the first account of AMPK-mediated regulation of SUR2A.
Assuntos
Isquemia Miocárdica/genética , Oxigênio/administração & dosagem , Proteínas Quinases/genética , Receptores de Sulfonilureias/genética , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Animais , Cardiotônicos/administração & dosagem , Cardiotônicos/metabolismo , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Canais KATP/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Isquemia Miocárdica/terapia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Ribonucleotídeos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Receptores de Sulfonilureias/metabolismoRESUMO
The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferatoractivated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Ligação a DNA/biossíntese , Proteínas Mitocondriais/biossíntese , Osteossarcoma/tratamento farmacológico , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Ribonucleotídeos/administração & dosagem , Fatores de Transcrição/biossíntese , Proteínas Quinases Ativadas por AMP/biossíntese , Aminoimidazol Carboxamida/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Resistance to chemotherapy is one of the major problems facing current cancer research. Enhancing tumor cell response to anticancer agents increases chemotherapeutic effectiveness. We have recently addressed this issue and reported on producing multifunctional nanoparticles (Fe3O4@SiO2(FITC)-FA/AICAR/DOX) aiming to overcome chemoresistance with synergetic effect of AICAR and DOX. In the present study, we demonstrated that these nanoparticles not only show enhanced cellular uptake and cytotoxic effect but can also show enhanced pro-apoptotic and anti-proliferative effects in five different tumor-derived cell lines (A549, HCT-116, HeLa, Jurkat and MIA PaCa-2). METHODS: The nanoparticles were examined by using flow cytometric analyses of apoptosis and cell cycle. In addition, we performed caspase-3 activity assay, which supported our flow cytometric data. Furthermore, we demonstrated the applicability of this approach in a variety of cancer types confirming the potential widespread utility of this approach. RESULTS: With the concept of co-delivery of AICAR and DOX in the nanoparticle formulation, the use of AICAR against survivin (BIRC5) sensitized cancer cells to DOX chemotherapy which resulted in effective cancer cell elimination. These result showed that combination therapy involving both a molecularly targeted therapy and chemotherapeutic agent has the ability to retain and enhance therapeutic efficacy. CONCLUSION: Fe3O4@SiO2(FITC)-FA/AICAR/DOX nanoparticles is superior to monotherapy via the synergetic effect of AICAR and DOX and also the nanoparticle formulation could overcome issues of toxicity with targeted therapy while maintaining the potent anticancer effects of AICAR and DOX. Graphical Abstract Apoptosis analysis of A549 cells by flow cytometry-based PE-annexin-V / 7-ADD double staining treated with low-dose (10 µg/ml) concentration of (1) Fe3O4@SiO2(FITC)-FA (2) Fe3O4@SiO2(FITC)-FA/AICAR, (3) Fe3O4@SiO2(FITC)-FA/DOX or (4) Fe3O4@SiO2(FITC)-FA/AICAR/DOX nanoparticles. Viable cells labelled with PE-annexin-V(-)/7-ADD(-), early apoptotic cells labelled with PE-annexin-V(+)/7-ADD(-) and apoptotic cells labelled with PE-annexin-V(+)/ 7-ADD(+) in flow cytometric graphics.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Proteínas Inibidoras de Apoptose/metabolismo , Nanopartículas/administração & dosagem , Ribonucleotídeos/administração & dosagem , Células A549 , Aminoimidazol Carboxamida/administração & dosagem , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Compostos Férricos/administração & dosagem , Ácido Fólico/administração & dosagem , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Terapia de Alvo Molecular/métodos , Dióxido de Silício/administração & dosagem , SurvivinaRESUMO
BACKGROUND: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-)). RESULTS: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle. CONCLUSIONS: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Glicemia/metabolismo , Ritmo Circadiano , Insulina/metabolismo , Músculo Esquelético/metabolismo , Tecido Adiposo/metabolismo , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Animais , Glicemia/análise , Peso Corporal , Feminino , Transportador de Glucose Tipo 4/metabolismo , Hexoquinase/metabolismo , Homeostase , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Fosfofrutoquinase-1 Muscular/metabolismo , RNA Mensageiro/metabolismo , Ribonucleotídeos/administração & dosagemRESUMO
A series of poly(ADP-ribose)polymerase (PARP)-1 inhibitors containing a novel scaffold, the 1H-thieno[3,4-d]imidazole-4-carboxamide moiety, was designed and synthesized. These efforts provided some compounds with relatively good PARP-1 inhibitory activity, and among them, 16l was the most potent one. Cellular evaluations indicated that the anti-proliferative activities of 16g, 16i, 16j and 16l against BRCA-deficient cell lines were similar to that of olaparib, while the cytotoxicities of 16j and 16l toward human normal cells were lower. In addition, ADMET prediction results indicated that these compounds might possess more favorable toxicity and pharmacokinetic properties. This study provides a basis for our further investigation.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Relação Estrutura-Atividade , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/síntese química , Aminoimidazol Carboxamida/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Poli(ADP-Ribose) Polimerase-1/químicaRESUMO
AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, which induces cytotoxic effect to several cancer cells. Its potential activity in prostate cancer cells and the underlying signaling mechanisms have not been extensively studied. Here, we showed that AICAR primarily induced programmed necrosis, but not apoptosis, in prostate cancer cells (LNCaP, PC-3 and PC-82 lines). AICAR's cytotoxicity to prostate cancer cells was largely attenuated by the necrosis inhibitor necrostatin-1. Mitochondrial protein cyclophilin-D (CYPD) is required for AICAR-induced programmed necrosis. CYPD inhibitors (cyclosporin A and sanglifehrin A) as well as CYPD shRNAs dramatically attenuated AICAR-induced prostate cancer cell necrosis and cytotoxicity. Notably, AICAR-induced cell necrosis appeared independent of AMPK, yet requiring reactive oxygen species (ROS) production. ROS scavengers (N-acetylcysteine and MnTBAP), but not AMPKα shRNAs, largely inhibited prostate cancer cell necrosis and cytotoxicity by AICAR. In summary, the results of the present study demonstrate mechanistic evidences that AMPK-independent programmed necrosis contributes to AICAR's cytotoxicity in prostate cancer cells.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeos/administração & dosagem , Aminoimidazol Carboxamida/administração & dosagem , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Masculino , Necrose/patologia , Neoplasias da Próstata/tratamento farmacológico , Resultado do TratamentoRESUMO
Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1ß or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.